cotransfection

Get More Out of Your Lentiviral Production

Posted on by Promega

fugene6_lvv_blogThis review is a guest blog by Amy Landreman, Product Specialist in Cellular Analysis at Promega Corporation.

Lentiviral vectors (LVV) have become a valuable research tool for delivering genetic content into a wide range of cell types. Commonly derived from the HIV-1 genome, LVV have the advantage of being able to infect both dividing and non-dividing cells. They can be particularly valuable for introducing genetic material into cell lines that are difficult to transfect using other methods and are also being used in gene therapy applications.

Unlike other gene delivery tools, transducing mammalian cells with LVV requires significant upfront effort since the LVV particles carrying the desired genetic content first need to be created. In general this involves co-transfecting a packaging cell line, such as HEK293T, with a set of three to four separate plasmids that encode the protein content required to generate the LVV particles: the transfer plasmid, which contains the transgene of interest, a packaging plasmid, and an envelope plasmid. After co-transfection, the packaging cell line is allowed to incubate for a couple of days during which time the LVV particles are produced and accumulating in the culture supernatant. The supernatant containing the recombinant LVV is then harvested and, following several concentration steps, the LVV particles are ready to be used for introducing the desired genetic content into the mammalian target cells. Continue reading “Get More Out of Your Lentiviral Production”

Screening for Drug-Drug Interactions with PXR and CYP450 3A4 Activation

Posted on by Promega

The pregnane X receptor (PXR) is a nuclear receptor known to regulate expression of cytochrome P450 (CYP450) drug-metabolizing enzymes (1). PXR has even been designated the “master xenosensor” due to its ability to upregulate cellular levels of a variety of drug-metabolizing enzymes in response to drugs and foreign chemicals. Elevated levels of CYP450 enzymes can elicit alterations in the pharmacokinetics of co-administered drugs, which can result in adverse drug-drug interactions (DDI) or diminished bioavailability. By assessing PXR activation and CYP450 enzyme induction early in the drug development process, many companies hope to reduce late-stage clinical failures and minimize the high costs associated with bringing a new drug to market.

Proportion of drugs metabolized by different CYPs

A paper by Shukla et al. (2) examined over 2,800 clinically used drugs for their ability to activate human PXR (hPXR) and rat PXR (rPXR), induce human cytochrome P450 3A4 enzyme (CYP3A4) at the cellular level, and bind hPXR at the protein level. Several studies have identified PXR as playing a key role in regulating the expression of CYP3A4, an enzyme involved in the metabolism of more than 50% of all drugs prescribed in humans. Since PXR activation and CYP3A4 induction have an impact on drug metabolism and pharmacokinetics, the authors wanted to obtain data that would be valuable in understanding structure-activity relationships (SARs), the connection between chemical structure and biological activity, when prioritizing new molecular entities (NMEs) for further in vitro and in vivo studies.

Continue reading “Screening for Drug-Drug Interactions with PXR and CYP450 3A4 Activation”